This invention relates to performance of antiviral drug susceptibility and resistance tests for identifying effective drug regimens for the treatment of viral infections. More particularly, the invention relates to methods and systems for performing antiviral drug susceptibility and resistance tests using robotics and software.
The term xe2x80x9cviral drug susceptibilityxe2x80x9d is generally understood to be the concentration of an antiviral agent at which a given percentage of viral replication is inhibited (e.g. the IC50 for an antiviral agent is the concentration at which 50% of virus replication is inhibited). Thus, a decrease in viral drug susceptibility is the hallmark that a mutant virus for which an antiviral agent is selected is becoming resistant to that antiviral drug. The term xe2x80x9cviral drug resistancexe2x80x9d is generally defined as a decrease in viral drug susceptibility in a given patient over time. In the clinical context, viral drug resistance is evidenced by the antiviral drug no longer being clinically effective in a patient.
Antiviral drug susceptibility and resistance tests are described in U.S. Pat. No. 5,837,464. A viral resistance assay in accordance with the antiviral drug susceptibility and resistance tests described therein comprises transfection, infection and plate reading steps.
Packaging host cells are plated into microtiter plates containing cell culture medium (e.g., f12:DMEM from Gibco 50:50 with added glutamine and without antibiotics) 48 hours prior to transfection at 2xc3x97104 cells per well. xe2x80x9cTransfectionxe2x80x9d as used herein means introducing DNA into a host cell so that the DNA is expressed, whether functionally or otherwise; the DNA may also replicate either as an extrachromosomal element or by chromosomal integration. One method which may be used for transformation of the packaging host cells is the calcium phosphate co-precipitation method of Graham and van der Eb (1973) Virology 52, 456-457. Alternative methods for transfection which may be used include the DEAE-dextran method, lipofection and biolistics as described in, for example, Kriegler (1990) Gene Transfer and Expression: A Laboratory Manual (Stockton Press).
In the calcium phosphate co-precipitation method, 5 to 10 mg each of the resistance test vector and the appropriate packaging expression vector(s), 100 microliters of 1M Calcium chloride and phosphate-buffered saline (PBS) are mixed to produce a precipitate. This precipitate is then added to the appropriate wells containing packaging host cells to produce resistance test vector host cells. The protease inhibitor drug(s) or medium is added to individual wells of the microtiter plate that contains packaging host cells at the time of their transfection, at an appropriate range of concentrations. Cell culture medium is added to wells to which drugs have not been added. The plates are lidded and the lidded, package host cell plates are placed in an incubator at 7% CO2, 37xc2x0 C. and 95% relative humidity for 24 to 48 hours.
Target host cell plates are made 24 hours prior to infection by adding 1.0xc3x97105 of cells in cell culture medium into the appropriate wells of a microtiter plate. The plates are lidded, then placed in an incubator at 7% CO2, 37xc2x0 C. and 95% relative humidity until they are infected. Just prior to infection, the target cell plates are removed from the incubator, the lid is removed and antiviral drugs or cell culture medium is added to the appropriate wells on the plate. The package host cell plates are removed from the incubator and de-lidded. The medium is removed and filtered through a 0.2 micron filter. The resulting viral is added to the appropriate wells in the target host cell plate. The target host cell plate is lidded and placed into an incubator at 7% CO2, 37xc2x0 C. and 95% relative humidity.
Twenty-four to forty-eight hours later, the target host cell plates are assayed for firefly luciferase activity as described in, for example, Ausubel et al. (1987) Current Protocols in Molecular Biology (Wiley-Interscience). The cell culture medium is removed from the wells, a lyse reagent is added and the plate is incubated at room temperature for 20 minutes. Luciferase substrate is added to the plate and the plate is read by a luminometer to determine the light output.
The antiviral drugs being added to the host cell plates are added at selected times depending upon the target of the antiviral drug. For example, in the case of HIV protease inhibitors, including amprenavir, nelfinavir, saquinavir, ritonavir, and indinavir, they are added to individual plates of packaging host cells at the time of their transfection with a resistance test vector, at an appropriate range of concentrations. HIV reverse transcriptase inhibitors, including AZT, ddI, ddC, d4T, 3TC. and nevaripine, are added to individual plates of target host cells at the time of infection by the resistance test vector viral particles, at a test concentration. The test concentration is selected from a range of concentrations which is typically between about 0.1 nM and about 100_M and more specifically for each of the following drugs: AZT, from about 1 nM to about 5_M; ddI, from about 1 nM to about 25_M; 3TC, from about 1 nM to about 50_M; d4T, from about 1 nM to about 25_M; and nevaripine, from about 1 nM to about 100_M.
Instrumentation for transfecting and infecting cells are known generally in the art, and most practitioners are familiar with the standard resource materials which describe their use and function. However, at present, the tools available to the researcher and clinician for performing antiviral drug susceptibility and resistance tests are inadequate. Manual methods for performing these tests are slow, tedious, and prone to human error. In addition, they are not easy to scale up, provide too low of a throughput for commercialization, and are labor intensive.
It is an object of this invention to provide automated systems and methods for evaluating the biological effectiveness of candidate drug compounds which act on specific viral genes and/or viral proteins particularly with respect to viral drug resistance and cross resistance.
Another object of this invention is to provide automated systems and methods for performing an assay for identifying and assessing the biological effectiveness of potential therapeutic compounds for treating viral diseases.
Yet another object of this invention is to provide automated systems and methods for performing antiviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of viral infections and screening candidate drugs for their capacity to inhibit selected viral sequences and/or viral proteins. The automated performance of the tests may be used for developing, for example, an optimal therapeutic regimen for treatment of HIV/AIDS. The systems and methods in accordance with the present invention may be used to automate the methods of performing drug susceptibility and resistance tests described in, for example, U.S. Pat. No. 5,837,464.
It is another object of this invention to provide automated systems and methods for performing antiviral drug susceptibility and resistance tests which use robotics and software, for example, to introduce a resistance test vector into a host cell, and to determine an expression or inhibition of the indicator gene product in a target host cell in the presence of an antiviral drug.
It is another object of this invention to provide systems and methods for performing the drug susceptibility and resistance test in a safe, standardized, rapid, precise and reliable manner for clinical and research application.
This and other objects of the invention will be apparent from the specification as a whole.
Objects of the present invention may be accomplished by configuring and adapting standard components in a novel manner to produce the following three automated apparatuses for performing the cell assay portion of antiviral drug susceptibility and resistance testing:
1) a Transfection Apparatus which may comprise
a) track-mounted robotic arm and controller,
b) robotic-friendly incubator,
c) automated high density storage unit,
d) two liquid handlers,
e) plate lid/de-lid station,
f) plate aspiration device,
g) barcode reader,
h) barcode label printer and application module,
i) HEPA air supply,
j) refrigerated hotels,
k) slave computer,
l) host computer and customized system control software, and
m) input/output interface box;
2) an Infection Apparatus which may comprise
a) track-mounted robotic arm and controller,
b) robotic-friendly incubator,
c) two automated high density storage units,
d) 96-channel pipettor,
e) plate lid/de-lid station,
f) plate filtration device,
g) barcode reader,
h) barcode label printer and application module,
i) HEPA air supply,
j) host computer and system control software, and
k) input/output interface box; and
(3) a Plate Reading System which may comprise
a) track-mounted robotic arm and controller,
b) two robotic-friendly incubators,
c) platewasher and dispenser,
d) reagent dispenser,
e) plate de-lid station,
f) plate reader,
g) barcode reader,
h) hotel,
i) host computer and system control software, and
j) input/output interface box.
The systems and methods in accordance with the present invention provide significant advantages over presently available methods for performing the assay in a safer, more affordable, more rapid and more reliable manner.